The object of this project is to use bacteriophages Phi29 and PRD1 and model systems to investigate the mechanism of linear DNA replication. One of the major gaps of our knowledge of DNA replication is the mechanism by which the 5'-ends of the linear duplex DNA are replicated. Since none of the known DNA polymerases can initiate de novo synthesis of DNA chains, the elucidation of the primer forming mechanism is crucial to our understanding of DNA replication. Recently, novel DNA polymerases have been discovered which utilize protein as primers for the initiation of DNA chain synthesis. This project is designed for development of cell-free systems for complete semiconserved replication of linear DNA molecules of Phi29 and PRD1. Using these systems, a model will be tested which involves circular intermediate of DNA-protein complex and which accounts for replication of displaced strand. Both in vitro and in vivo crosslinking of DNA-protein complex will be investigated using electronmicroscopy and immunochemical methods. In addition, to understand details of protein-priming mechanism, it is proposed to isolate various mutants of DNA polymerases and DNA terminal proteins for both Phi29 and PRD1. It is proposed that mutant proteins be used for investigation of protein-protein, protein-DNA interactions and for structure-function analysis. If, as we believe, the basic mechanism of DNA replication may prove to be applicable to more complex systems, information obtained from these studies of bacterial viruses will undoubtedly contribute to our understanding of DNA replication in higher organisms.